Arrhythmias are a manifestation of acquired or congenital cardiac conduction system disease affecting at least 5% of the US population. They are an often lethal complication of myocardial infarction and cardiovascular disease, and the major cause of morbidity and death in congenital heart disease patients. Normal conduction system function is critical for prenatal survival. Tbx3 is a transcription factor expressed in the conduction system myocardium. Development and homeostasis of the sinoatrial and atrioventricular nodes (SAN, AVN) are extremely sensitive to Tbx3: embryonic or adult deficiency in mice causes a variety of arrhythmias and sudden death. Recent studies confirm the importance of TBX3 in human conduction system function. We have discovered that Tbx3 regulates alternative splicing, transcription initiation and termination. These alternative RNA processing events are the most important contributors to tissue-specific gene expression. Our data indicate that Tbx3-regulated alternative RNA processing is critical for conduction system development and homeostasis. In this proposal, we will identify Tbx3 target RNAs in the developing conduction system and underlying regulatory mechanisms. Discover and compare RNA signatures of control and Tbx3 mutant embryonic SAN, AV canal (contains AVN progenitors), atrial and ventricular working myocardium and thus determine how Tbx3 regulates the differentiation of different functional compartments within the conduction system. We will determine the functional impact of Tbx3-regulated alternative mRNAs. We will determine how TBX3 mutations that cause Ulnar-mammary syndrome and arrhythmias in humans affect its RNA processing functions and protein interactions. This proposal continues our detailed investigation into roles of Tbx3 during early cardiac morphogenesis and how this crucial factor regulates conduction system development. This topic is highly relevant to understanding the pathogenesis of human congenital and acquired arrhythmias. We develop and employ a powerful combination of approaches to delve into the molecular mechanisms underlying differentiation of highly specialized CS tissues. We have engaged a group of expert collaborators to achieve the aims of this proposal and apply them to the larger questions of conduction system homeostasis and regeneration in the future.